LOCUS       sequence-325773-        7899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  heat shock-induced expression of Cas9 in C. elegans.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7899)
  AUTHORS   Froehlich JJ, Uyar B, Herzog M, Theil K, Glazar P, Akalin A, 
            Rajewsky N
  TITLE     Parallel genetics of regulatory sequences using scalable genome 
            editing in vivo.
  JOURNAL   Cell Rep. 2021 Apr 13;35(2):108988. doi: 
            10.1016/j.celrep.2021.108988.
  PUBMED    33852857
REFERENCE   2  (bases 1 to 7899)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Rep.";
            date: "2021-04-13"; volume: "35(2):108988. doi"; pages: " 
            10.1016/j.celrep.2021.108988"
COMMENT     Sequence Label: pJJF152_Phsp_Cas9
FEATURES             Location/Qualifiers
     source          1..7899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        452..757
                     /label=hsp16-48 promoter
                     /note="promoter for the C. elegans heat shock protein 16-48
                     gene (Russnak and Candido, 1985)"
     CDS             join(764..2839,2891..4918)
                     /codon_start=1
                     /product="S. pyogenes Cas9, codon optimized for C. elegans 
                     with a synthetic intron"
                     /label=Cas9
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4931..4951
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5678..5694)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5678..5694)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5691..5713)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5702..5718
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5726..5732,5733..5750,5751..5756))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5771..5792
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5909..5926)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6080..6668)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6160..6179)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6839..7630,7631..7699))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7462..7481
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7700..7804)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7872..7890
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cactagtatg attgtagttt
      421 gaagatttca caattagagt gaatgttgtt tggttcggtt ttgtcactgt atttatactc
      481 atttccacct ttttctagaa cattcgagct gcttcttgca aaaggagggc gactcacatt
      541 cagaacattg agaaatagtg tgcgtactga agaaacccag atactttttc aatctgcgtc
      601 tctttgcacc tatggggtgt attttgaaat gaatgcatct aggaccttct agaacattct
      661 aaacggctgc aggatacggg tatataagcc aatcgtgttc agaggaaacc aatacacttt
      721 gttcaagtgc ttactgttca ttctctaaac ttcaagagct agcatggaca agaagtactc
      781 catcggactc gacatcggaa ccaactccgt cggatgggcc gtcatcaccg acgagtacaa
      841 ggtcccatcc aagaagttca aggtcctcgg aaacaccgac cgtcactcca tcaagaagaa
      901 cctcatcgga gccctcctct tcgactccgg agagaccgcc gaggccaccc gtctcaagcg
      961 taccgcccgt cgtcgttaca cccgtcgtaa gaaccgtatc tgctacctcc aggagatctt
     1021 ctccaacgag atggccaagg tcgacgactc cttcttccac cgtctcgagg agtccttcct
     1081 cgtcgaggag gacaagaagc acgagcgtca cccaatcttc ggaaacatcg tcgacgaggt
     1141 cgcctaccac gagaagtacc caaccatcta ccacctccgt aagaagctcg tcgactccac
     1201 cgacaaggcc gacctccgtc tcatctacct cgccctcgcc cacatgatca agttccgtgg
     1261 acacttcctc atcgagggag acctcaaccc agacaactcc gacgtcgaca agctcttcat
     1321 ccagctcgtc cagacctaca accagctctt cgaggagaac ccaatcaacg cctccggagt
     1381 cgacgccaag gccatcctct ccgcccgtct ctccaagtcc cgtcgtctcg agaacctcat
     1441 cgcccagctc ccaggagaga agaagaacgg actcttcgga aacctcatcg ccctctccct
     1501 cggactcacc ccaaacttca agtccaactt cgacctcgcc gaggacgcca agctccagct
     1561 ctccaaggac acctacgacg acgacctcga caacctcctc gcccagatcg gagaccagta
     1621 cgccgacctc ttcctcgccg ccaagaacct ctccgacgcc atcctcctct ccgacatcct
     1681 ccgtgtcaac accgagatca ccaaggcccc actctccgcc tccatgatca agcgttacga
     1741 cgagcaccac caggacctca ccctcctcaa ggccctcgtc cgtcagcagc tcccagagaa
     1801 gtacaaggag atcttcttcg accagtccaa gaacggatac gccggataca tcgacggagg
     1861 agcctcccag gaggagttct acaagttcat caagccaatc ctcgagaaga tggacggaac
     1921 cgaggagctc ctcgtcaagc tcaaccgtga ggacctcctc cgtaagcagc gtaccttcga
     1981 caacggatcc atcccacacc agatccacct cggagagctc cacgccatcc tccgtcgtca
     2041 ggaggacttc tacccattcc tcaaggacaa ccgtgagaag atcgagaaga tcctcacctt
     2101 ccgtatccca tactacgtcg gaccactcgc ccgtggaaac tcccgtttcg cctggatgac
     2161 ccgtaagtcc gaggagacca tcaccccatg gaacttcgag gaggtcgtcg acaagggagc
     2221 ctccgcccag tccttcatcg agcgtatgac caacttcgac aagaacctcc caaacgagaa
     2281 ggtcctccca aagcactccc tcctctacga gtacttcacc gtctacaacg agctcaccaa
     2341 ggtcaagtac gtcaccgagg gaatgcgtaa gccagccttc ctctccggag agcagaagaa
     2401 ggccatcgtc gacctcctct tcaagaccaa ccgtaaggtc accgtcaagc agctcaagga
     2461 ggactacttc aagaagatcg agtgcttcga ctccgtcgag atctccggag tcgaggaccg
     2521 tttcaacgcc tccctcggaa cctaccacga cctcctcaag atcatcaagg acaaggactt
     2581 cctcgacaac gaggagaacg aggacatcct cgaggacatc gtcctcaccc tcaccctctt
     2641 cgaggaccgt gagatgatcg aggagcgtct caagacctac gcccacctct tcgacgacaa
     2701 ggtcatgaag cagctcaagc gtcgtcgtta caccggatgg ggacgtctct cccgtaagct
     2761 catcaacgga atccgtgaca agcagtccgg aaagaccatc ctcgacttcc tcaagtccga
     2821 cggattcgcc aaccgtaacg taagtttaaa catgatttta ctaactaact aatctgattt
     2881 aaattttcag ttcatgcagc tcatccacga cgactccctc accttcaagg aggacatcca
     2941 gaaggcccag gtctccggac agggagactc cctccacgag cacatcgcca acctcgccgg
     3001 atccccagcc atcaagaagg gaatcctcca gaccgtcaag gtcgtcgacg agctcgtcaa
     3061 ggtcatggga cgtcacaagc cagagaacat cgtcatcgag atggcccgtg agaaccagac
     3121 cacccagaag ggacagaaga actcccgtga gcgtatgaag cgtatcgagg agggaatcaa
     3181 ggagctcgga tcccagatcc tcaaggagca cccagtcgag aacacccagc tccagaacga
     3241 gaagctctac ctctactacc tccagaacgg acgtgacatg tacgtcgacc aggagctcga
     3301 catcaaccgt ctctccgact acgacgtcga ccacatcgtc ccacagtcct tcctcaagga
     3361 cgactccatc gacaacaagg tcctcacccg ttccgacaag aaccgtggaa agtccgacaa
     3421 cgtcccatcc gaggaggtcg tcaagaagat gaagaactac tggcgtcagc tcctcaacgc
     3481 caagctcatc acccagcgta agttcgacaa cctcaccaag gccgagcgtg gaggactctc
     3541 cgagctcgac aaggccggat tcatcaagcg tcagctcgtc gagacccgtc agatcaccaa
     3601 gcacgtcgcc cagatcctcg actcccgtat gaacaccaag tacgacgaga acgacaagct
     3661 catccgtgag gtcaaggtca tcaccctcaa gtccaagctc gtctccgact tccgtaagga
     3721 cttccagttc tacaaggtcc gtgagatcaa caactaccac cacgcccacg acgcctacct
     3781 caacgccgtc gtcggaaccg ccctcatcaa gaagtaccca aagctcgagt ccgagttcgt
     3841 ctacggagac tacaaggtct acgacgtccg taagatgatc gccaagtccg agcaggagat
     3901 cggaaaggcc accgccaagt acttcttcta ctccaacatc atgaacttct tcaagaccga
     3961 gatcaccctc gccaacggag agatccgtaa gcgtccactc atcgagacca acggagagac
     4021 cggagagatc gtctgggaca agggacgtga cttcgccacc gtccgtaagg tcctctccat
     4081 gccacaggtc aacatcgtca agaagaccga ggtccagacc ggaggattct ccaaggagtc
     4141 catcctccca aagcgtaact ccgacaagct catcgcccgt aagaaggact gggacccaaa
     4201 gaagtacgga ggattcgact ccccaaccgt cgcctactcc gtcctcgtcg tcgccaaggt
     4261 cgagaaggga aagtccaaga agctcaagtc cgtcaaggag ctcctcggaa tcaccatcat
     4321 ggagcgttcc tccttcgaga agaacccaat cgacttcctc gaggccaagg gatacaagga
     4381 ggtcaagaag gacctcatca tcaagctccc aaagtactcc ctcttcgagc tcgagaacgg
     4441 acgtaagcgt atgctcgcct ccgccggaga gctccagaag ggaaacgagc tcgccctccc
     4501 atccaagtac gtcaacttcc tctacctcgc ctcccactac gagaagctca agggatcccc
     4561 agaggacaac gagcagaagc agctcttcgt cgagcagcac aagcactacc tcgacgagat
     4621 catcgagcag atctccgagt tctccaagcg tgtcatcctc gccgacgcca acctcgacaa
     4681 ggtcctctcc gcctacaaca agcaccgtga caagccaatc cgtgagcagg ccgagaacat
     4741 catccacctc ttcaccctca ccaacctcgg agccccagcc gccttcaagt acttcgacac
     4801 caccatcgac cgtaagcgtt acacctccac caaggaggtc ctcgacgcca ccctcatcca
     4861 ccagtccatc accggactct acgagacccg tatcgacctc tcccagctcg gaggagactc
     4921 ccgtgccgac ccaaagaaga agcgtaaggt ctgacctagg gtccaattac tcttcaacat
     4981 ccctacatgc tctttctccc tgtgctccca ccccctattt ttgttattat caaaaaactt
     5041 ctcttaattt ctttgttttt tagcttcttt taagtcacct ctaacaatga aattgtgtag
     5101 attcaaaaat agaattaatt cgtaataaaa agtcgaaaaa aattgtgctc cctcccccca
     5161 ttaataataa ttctatccca aaatctacac aatgttctgt gtacacttct tatgtttttt
     5221 acttctgata aatttttttg aaacatcata gaaaaaaccg cacacaaaat accttatcat
     5281 atgttacgtt tcagtttatg accgcaattt ttatttcttc gcacgtctgg gcctctcatg
     5341 acgtcaaatc atgctcatcg tgaaaaagtt ttggagtatt tttggaattt ttcaatcaag
     5401 tgaaagttta tgaaattaat tttcctgctt ttgctttttg gggtttcccc tattgtttgt
     5461 caagatttcg aggacggcgt ttttcttgct aaaatcacaa gtattgatga gcacgatgca
     5521 agaaagatcg gaagaaggtt tgggtttgag gctcagtgga aggtgagtag aagttgataa
     5581 tttgaaagtg gagtagtgtc tatggggttt ttgccttaaa tgacagaata cattcccaat
     5641 ataccaaaca taactgttta agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5701 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5761 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5821 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5881 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5941 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6001 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6061 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6121 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6181 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6241 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6301 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6361 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6421 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6481 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6541 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6601 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6661 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6721 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6781 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6841 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6901 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6961 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7021 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7081 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7141 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7201 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7261 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7321 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7381 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7441 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7501 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7561 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7621 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7681 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7741 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7801 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7861 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
